Wednesday, November 12, 2008

Q1. what is the source of energy for light production in the firefly ?”

1. ATP2. GTP3. phosho creatine4. NADH

The answer is ATP….

Let me start the discussion with this question …..“DO YOU THINK THAT THE QUESTION IS RELEVANT TO US ?”

If you think this question is irrelevant , then go through the following discussion & then you ll understand why these AIIMSpeople want us to know about the metabolic machinery of this humble organism.

The firefly (Photinus pyralis) contains an enzyme called LUCIFERASE. This enzyme acts on its substrate – luciferin in the presence of ATP and calcium. The reaction produces visible light. This reaction has been exploited by the scientific community for research on

MDR TB is characterized by the presence of resistance to the two drugsIsoniazide …….. due to mutations of genes Kat G & INH ARifampicin……..due the mutations in the gene rpo B gene

To the mycobacteria cultured from the patient`s clinical samples we add adeno virus carrying the FFLUX gene (which codes for luciferase). Within a few minutes the mycobacteria exhibit bioluminescence i.e they emit light. Now add the antibiotic , say INH. If the mycobacteria are sensitive , they soon become non viable & their metabolic machinery is paralysed and hence ATP is depleted. In the absence of ATP , the light production is reduced. In fact the degree of reduction of the bio luminescence can be taken as an indicator of the potency of the drug .If the strains are resistant , there is NO reduction in light production

PYRO SEQUENCING

This is a type of DNA sequencing.Take the template DNA which is to be sequenced. To this we add luciferase, luciferin, calcium , DNA polymerase, ATP sulfurylaseThen we add the deoxy nucleotides (A, C , G , T) one by one and wash them off.For instance let the d-NTP in x position of the template be A. When we add A, G , C there will be no reaction. But when we add T , base pairing occurs , releasing a PYRO PHOSPHATE, which is acted upon by ATP sulfurylase to produce ATP.When there is ATP , luciferase is activated and there is luminescence. So we know that the template contains the d NTP which is complementary to the one that we added.in the same way the entire NA can be sequenced

STUDYING METABOLIC PATHWAYS

Coenorhabditis elegans (a nematode) was the FIRST MULTICELLULAR ORGANISM FOR WHICH THE ENTIRE GENOME WAS SEQUENCED.

An interesting aspect is that its cuticular layer is transparent. We can add the luciferase gene to its genome with the help of adeno or lenti virus. So whenever its metabolic machinery is activated an ATP is produced, light is emitted.Likewise whenever a metabolic poison is given the ‘light is switched off’The degree of bio luminescence is proportional to the amount of ATP present

STUDYING TRANSCRIPTIONAL ACTIVITIES

To study the transcriptional activity of a gene , incorporate the luciferase gene close to the index gene `s promoterWhenever there is transcription, luciferase is also activated and light is emitted

TUMORIGENESIS MODELS

The tumor cells tagged with luciferase gene become luminescent and thereby their growth and susceptibility to chemo therapeutic agents can be studied in vivo within mouse models

OTHERS

Luciferase gene is also used in forensic medicine - detect blood and in studying specific cell lines

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